1. Field of the Invention
The present invention relates to novel polyhydroxyalkanoate (PHA), as well as a method of producing such a novel PHA by utilizing microorganisms.
2. Related Background Art
Synthetic polymers derived from petroleum have been used as plastics etc. for a long time. Recently, the treatment of the used plastics has become one of serious social problems. These synthetic polymers have advantages of hard-to-decompose have been used in the place of metal or glass materials. On mass consumption and mass disposal, however, this feature of hard-to-decompose makes them accumulated in waste-disposal facilities, or when they are burned, it causes increased carbon dioxide exhaust, and harmful substances such as dioxin and endocrine-disruptors may be generated to cause environmental pollution.
On the other hand, polyesters produced by microorganisms (hereinafter referred to as xe2x80x9cmicrobial polyestersxe2x80x9d) can be biologically degraded to be incorporated in a natural recycling system. Thus they would not remain in natural environment without causing pollution, in contrast to the numerous usual synthetic polymer compounds. Furthermore, since the biodegradability dispenses with incinerating treatment, microbial polyesters are effective from the standpoint of the prevention of air pollution and global warming, and usable as plastics to maintain the environment. In addition, their potential as soft materials for medical use has been investigated (Japanese Patent Application Laid-Open No. 5-159, Nos. 6-169980, 6-169988, 6-225921, etc.).
Heretofore, various bacteria have been reported to produce and accumulate PHB or copolymers of other hydroxyalkanoic acids in the cells (Handbook of Biodegradable Plastics, ed. by Biodegradable Plastics Society, published by N.T.S., p. 178-197 (1995)). Microbial PHA thus obtained is known to have various compositions and structures depending on the class of microorganisms used, medium composition, culture conditions, etc. during production, and many studies related to the control of composition and structure of PHA products have been conducted to improve PHA properties.
For example, Alcaligenes eutropus H16 ATCC No. 17699 and its mutants can produce copolymers of 3-hydroxybutyric acid (3HB) and 3-hydroxyvaleric acid (3HV) at a various composition ratio by varying carbon sources during culture (Published Japanese Translation of PCT International Publication Nos. 6-15604, 7-14352, 8-19227, etc.).
Japanese Patent No. 2642937 discloses that Pseudomonas oleovorans ATCC29347, when given acyclic aliphatic hydrocarbons as a carbon source, produces PHA having a monomer unit of 3-hydroxyalkanoate of 6 to 12 carbon atoms.
Japanese Patent Application Laid-Open No. 5-74492 discloses the method comprising contacting a microorganism of Methylobacterium sp., Paracoccus sp., Alcaligenes sp., or Pseudomonas sp. with a primary alcohol of 3 to 7 carbon atoms, thereby allowing to produce a copolymer of 3HB and 3HV.
Japanese Patent Application Laid-Open Nos. 5-93049 and 7-265065 disclose that Aeromonas caviae can produce, by using oleic acid and olive oil as carbon sources, a binary copolymer of 3HB and 3-hydroxyhexanoic acid (3HHx).
Japanese Patent Application Laid-Open No. 9-191893 discloses that Comamonas acidovorans IFO13852 can produce, by using gluconic acid and 1,4-butanediol as a carbon source, a polyester having monomer units of 3HB and 4-hydroxybutyric acid.
Furthermore, certain microorganisms has been reported to produce PHA having various substituents such as groups derived from unsaturated hydrocarbons, ester group, allyl group, cyano group, nitro group, groups derived from halogenated hydrocarbon, and epoxide. Thus, there have been started several attempts to improve the properties of microbial PHA by using such a technique. Examples of microbial polyester having such substituents are described in FEMS Microbiology Letters, 128 (1995) p.219-228, in detail. Makromol. Chem., 191, 1957-1965, 1990, Macromolecules, 24, 5256-5260, 1991, and Chirality, 3, 492-494, 1991 report that Pseudomonas oleovorans produces PHA comprising a monomer unit of 3-hydroxy-5-phenylvaleric acid (3HPV), and changes in polymer properties probably due to the presence of the monomer unit of 3HPV.
As stated above, microbial PHA of various compositions/structures can be obtained by varying the microorganism, medium composition, culture conditions, etc. for polymer production. Their physical properties, however, are still insufficient for plastics. In order to further extend the application field, it is important to investigate more extensively the improvement of properties, and it is, therefore, essential to develop and search PHA made of structurally various monomer units, methods of producing them, as well as microorganisms capable of efficiently producing the desired PHA.
On the other hand, those PHA having introduced substituents in the side chains as described above, can be expected to be developed as xe2x80x9cfunctional polymerxe2x80x9d having useful functions and properties by selecting the substituent to be introduced according to the desired properties, etc. It is also important to develop and search PHA satisfying both functionality and biodegradability, methods of producing them, as well as microorganisms capable of efficiently producing desired PHA.
One example of such PHA having a substituent introduced in side chains is PHA having phenoxy in side chains.
For example, Macromol. Chem. Phys., 195, 1655-1672 (1994) reports that Pseudomonas oleovorans produces PHA containing units of 3-hydroxy-5-phenoxyvaleric acid and 3-hydroxy-9-phenoxynonanoic acid, from 11-phenoxyundecanoic acid.
Macromolecules, 29, 3432-3435 (1996) also reports that Pseudomonas oleovorans can be used to produce PHA containing 3-hydroxy-4-phenoxyburyric acid and 3xe2x80x98-hydroxy-6-phenoxyhexanoic acid units from 6-phenoxyhexanoic acid, PHA containing 3-hydroxy-6-phenoxyhexanoic acid and 3-hydroxy-8-phenoxyoctanoic acid units from 8-phenoxyoctanoic acid, and PHA containing 3-hydroxy-5-phenoxyvaleric acid and 3-hydroxy-7-phenoxyheptanoic acid units from 11-phenoxyunndecanoic acid. The polymer yield is as follows.
Furthermore, Can. J. Microbiol., 41, 32-43 (1995) reports that when given octanoic acid and p-cyanophenoxyhexanoic acid or p-nitrophenoxyhexanoic acid as substrates, Pseudomonas oleovorans ATCC29347 or Pseudomonas putida KT2442 can produce PHA containing a monomer unit of 3-hydroxy-p-cyanophenoxyhexanoic acid or 3-hydroxy-p-nitrophenoxyhexanoic acid.
Japanese Patent No. 2989175 describes a homopolymer consisting of 3-hydroxy-5-(monofluorophenoxy)pentanoate (3H5(MFP)P) unit or 3-hydroxy-5-(difluorophenoxy)pentanoate (3H5(DFP)P) unit, a copolymer containing at least one of 3H5(MFP)P unit and 3H5(DFP)P unit, Pseudomonas putida which can produce such polymers; and a method of producing the above polymers by using a Pseudomonas sp.
Such productions are conducted by xe2x80x9c2-step culturexe2x80x9d described below. Culture period: step 1 - 24 hours; step 2 - 96 hours.
Substrates in each step and polymers obtained are as follows.
(1) Polymer obtained: 3H5(MFP)P homopolymer
Substrates in step 1: citric acid, yeast extract
Substrates in step 2: monofluorophenoxyundecanoic acid
(2) Polymer obtained: 3H5(DFP)P homopolymer
Substrates in step 1: citric acid, yeast extract
Substrates in step 2: difluorophenoxyundecanoic acid
(3) Polymer obtained: 3H5(MFP)P copolymer
Substrates in step 1: octanoic or nonanoic acid, yeast extract
Substrates in step 2: monofluorophenoxyundecanoic acid
(4) Polymer obtained: 3H5(MFP)P homopolymer
Substrates in step 1: octanoic or nonanoic acid, yeast extract
Substrates in step 2: difluorophenoxyundecanoic acid
It describes that the microorganism can assimilate substituted aliphatic acids of a medium chain length to produce a polymer having phenoxy group substituted with 1 to 2 fluorine atoms at the end of a side chain, and such a polymer has stereoregularity and water repellency while keeping a high melting point and a good processibility.
It has been reported a PHA containing a cyclohexyl group in its monomer unit is expected to exhibit polymer properties differing from a PHA containing an usual aliphatic hydroxyalkanoic acid as a unit, as well as its production by Pseudomonas oleovorans (Macromolecules, 30, 1611-1615 (1997)).
According to this report, Pseudomonas oleovorans is cultured in a medium containing nonanoic acid (hereinafter referred to as NA), and 4-cyclohexylbutyric acid (hereinafter referred to as CHBA) or 5-cyclohexylvaleric acid (hereinafter referred to as CHVA) to obtain PHA made of a cyclohexyl-containing unit and a unit derived from nonanoic acid (each proportion is unknown).
By varying the ratio of CHBA to NA under the conditions that the total concentration of substrates is 20 mM, the results shown in Table 2 were obtained. In Table 2, CDW: Cell mass (dry weight) (mg/L); PDW: polymer mass (dry weight) (mg/L); and Yield: PDW/CDW (%)
In this case, however, the polymer yield per culture (w/v) was insufficient, and nonanoic acid-derived aliphatic hydroxyalkanoic acid units were present in the resultant PHA.
As described above, to produce PHA having various introduced substituents in the side chain, as with the above Pseudomonas oleovorans, an alkanoate having a substituent to be introduced has been utilized not only as a polymer raw material but also as a carbon source for growth.
Such a method to utilize an alkanoate having a substituent to be introduced into the polymer, not only as a raw material for the polymer but also as a carbon source for growth expects to supply the carbon source and energy source as the acetyl-CoA formed by xcex2-oxidation of the alkanoate. In such a method, however, acetyl-CoA would not be formed by xcex2-oxidation unless the substrate has a certain chain length, so that there is a serious problem that the alkanoate available as the substrate for PHA is limited. In general, xcex2-oxidation generates a new substrate, of which chain length is shorter by two methylene units at a time, and they are incorporated as the monomer units of PHA, synthesized PHA is often a copolymer consisting of monomer units each differing by two methylene chains in the chain length. In the foregoing report, the produced polymer is a copolymer consisting of three monomer units, that is, 3-hydroxy-8-phenoxyoctanoic acid derived from the substrate 8-phenoxyoctanoic acid, 3-hydroxy-6-phenoxyhexanoic acid and 3-hydroxy-4-phenoxybutyric acid being metabolic by-products. Thus, PHA consisting of a single monomer unit is hard to obtain by this method. Furthermore, in the method depending on the acetyl-CoA formed by xcex2-oxidation as the carbon and energy source, there are such problems as slow growth rate of the microorganism, slow synthesis of PHA, and low yield of PHA.
Thus, usually the microorganism is grown in a medium containing a medium-length aliphatic acid such as octanoic acid and nonanoic acid, etc. as a carbon source for growth in addition to the alkanoate having a substituent to be introduced, and then PHA is extracted.
The PHA produced by the above method, however, contains monomer units having a substituent to be introduced and monomer units derived from the carbon source for growth (for example, 3-hydroxyoctanoic acid and 3-hydroxynonanoic acid). The polymer of such a medium chain length (mcl) monomer unit is adhesive at ambient temperature, and, when mixed with the desired PHA, significantly lowers the glass transition point (Tg). Thus, to obtain a polymer being solid at ambient temperature, contamination of mcl-monomer units is undesirable. In addition, the presence of heterogeneous side chains is known to interfere with intramolecular or intermolecular interactions due to the side chain structure, and significantly affects crystallinity and orientation. In order to improve the polymer properties and endowment of functions, a mixture of such mcl-monomer units is a serious problem. One means to solve this problem is to add a purification step to separate and remove such xe2x80x9cunintendedxe2x80x9d polymers of mcl-monomer units derived from the carbon source for growth and to obtain PHA consisting only of a monomer unit having a specific substituent. Nevertheless, operations become troublesome and a significant decrease of the yield is inevitable. A more important problem is the fact that, if the intended monomer units form a copolymer with the unintended monomer units, it is very difficult to remove the unintended monomer units only. In particular, when the PHA containing monomer units having such groups as the groups derived from unsaturated hydrocarbons, ester groups, aryl group, cyano group, nitro group, groups derived from halogenated hydrocarbons and epoxide as side chain structure, mcl-monomer units often form a copolymer with the intended monomer unit, so it is very difficult to remove mcl-monomer units after the PHA synthesis.
The present invention can solve the above problems. The object of the present invention is to provide a PHA containing monomer units of various structures having substituents useful for device materials, medical materials, etc. in the side chains. Another object of the present invention is to provide a method of producing such a PHA by utilizing microorganisms, especially a method of producing PHA with little contamination of monomer units and in a high yield. The other object of the present invention is to provide novel PHA consisting only of the desired monomer units without contamination of unintended monomer units, as well as a method of producing such a PHA by utilizing microorganisms.
In order to solve the above problems, especially to develop PHA having substituted or unsubstituted phenoxy group, phenyl group and cyclohexyl group in the side chains, being useful as device materials, medical materials, etc., the present inventor have extensively searched for novel microorganisms capable of producing and accumulating PHA in the cell, and a method of producing the desired PHA by utilizing novel microorganisms.
Further, to develop a method of obtaining efficiently the desired PHA without mixing of unintended monomer units, the present inventors made extensive study and found that by culturing the microorganism in a medium supplemented with yeast extract in addition to an alkanoate having a desired atomic group, it is possible to produce selectively only the desired PHA without being mixed with unintended monomer units or with reduced incorporation of unintended monomer units, then completed the present invention.
Thus, the method of producing novel PHA of the present invention is characterized by culturing a microorganism in a culture medium containing an alkanoate and yeast extract, which microorganism is capable of producing the object PHA by utilizing the alkanoate in the medium as a low material. In particular, the method of producing PHA of the present invention can be carried out in accordance with the embodiments described below.
According to one aspect of the present invention, there is provided a polyhydroxyalkanoate comprising one or more of monomer units represented by Formula (1), 
where R is at least one selected from the group represented by any one of Formulas (2), (3) and (4); 
in Formula (2), R1 is selected from the group consisting of hydrogen atom (H), halogen atom, xe2x80x94CN, xe2x80x94NO2, xe2x80x94CF3, xe2x80x94C2F5 and xe2x80x94C3F7, and q is an integer of 1 to 8;
in Formula (3), R2 is selected from the group consisting of hydrogen atom (H), halogen atom, xe2x80x94CN, xe2x80x94NO2, xe2x80x94CF3, xe2x80x94C2F5 and xe2x80x94C3F7, and r is an integer of 1 to 8;
in Formula (4), R3 is selected from the group consisting of hydrogen atom (H), halogen atom, xe2x80x94CN, xe2x80x94NO2, xe2x80x94CF3, xe2x80x94C2F5 and xe2x80x94C3F7, and s is an integer of 1 to 8;
provided that following R is not selected:
when one kind of R is selected:
R being R1=H and q=2, R being R1=H and q=3 in Formula (2),
R being R2=halogen and r=2, or R being R2=xe2x80x94CN and r=3 and
R being R2=xe2x80x94NO2 and r=3 in Formula (3);
when two kinds of R are selected:
a combination of R being R1=H and q=3 and 5 respectively in Formula (2),
a combination of R being R2=H and r=2 and 4 respectively,
a combination of R being R2=H and r=2 and 6 respectively, and
a combination of R being R2=halogen and r=2 and 4 respectively in Formula (3);
when three kinds of R are selected:
a combination of R being R1=H and q=3, 5 and 7 respectively in Formula (2),
a combination of R being R2=H and r=1, 3 and 5 respectively, and a combination of R being R2=H and r=2, 4 and 6 respectively in Formula (3).
According to another aspect of the present invention, there is provided a process of producing a polyhydroxyalkanoate comprising the step of:
culturing a microorganism in a culture medium containing a raw material alkanoate and an yeast extract, wherein the microorganism produces a polyhydroxyalkanoate utilizing the alkanoate.
The present invention provides a method for producing polyhydroxyalkanoate, which uses xcfx89-substituted-straight-chain alkanoic acid, of which terminal of a chain is substituted by any one of 6-carbon ring atomic group of a substituted or unsubstituted phenyl group, a substituted or unsubstituted phenoxy group, and a substituted or unsubstituted cyclohexyl group, as the material and also which contains corresponding xcfx89-substituted-3-hydroxy-alkanoic acid as the monomer units, and also provides microorganisms suitable for selective production of polyhydroxyalkanoate having 6-carbon ring atomic group in the terminal of these side chains. Various polyhydroxyalkanoate, of which production by microorganisms becomes first possible according to the present invention, in an inorganic culture medium containing the yeast extract and the xcfx89-substituted-straight-chain alkanoic acid as the material, a microorganism belonging to the genus Pseudomonas, for example, is cultured to work on the xcfx89-substituted-straight-chain alkanoic acid as the material allowing an efficient production. Therefore, polyhydroxyalkanoate useful as a functional polymer having biodegradability can be expected application thereof to various fields such as a device material and a material for a medical treatment.